Material and method to screen proteasome stimulators

ABSTRACT

The present disclosure relates to a group of peptide compounds and their use in identifying molecules that stimulate proteasome or immunoproteasome are disclosed herein. Composition matters and methods of uses are within the scope of this disclosure.

CROSS-REFERENCE TO RELATED APPLICATION

This Application is a divisional application of U.S. patent applicationSer. No. 16/128,697, filed Sep. 12, 2018, which relates to and claimsthe benefit of U.S. provisional application 62/559,076, filed on Sep.15, 2017. The contents of which are expressly incorporated herein byreference in its entirety into this present disclosure.

STATEMENT OF SEQUENCE LISTING

A computer-readable form (CRF) document of the Sequence Listing issubmitted with this application. The document, entitled67888-02_Seq_Listing_ST25_txt, is generated on Sep. 29, 2020. Applicantstates that the content of the computer-readable form is the same andthe information recorded in computer readable form is identical to thewritten sequence listing.

FIELD OF INVENTION

This disclosure relates to a fluorescent probe that helps identifyingmolecules that stimulate proteasome or immunoproteasome. Particularly, agroup of FRET-reporter peptides are used to identify at least two modesof action of stimulating proteasome core particle 20S CP to degradeproteins, including gate opener and chymotrypsin-like activity of 20SCP.

BACKGROUND

One of the most basic, essential cellular processes is the degradationof proteins. This is performed through two main pathways. Proteins canbe shuttled to the lysosome or degraded by a large enzyme complex knownas the proteasome.¹ The 26S proteasome is composed of the 19S regulatoryparticle (19S RP) and the 20S core particle (20S CP), FIG. 1.² For aprotein to be degraded by the 26S proteasome, it needs to be tagged withubiquitin. This tag is then recognized by a subunit of the 19Sregulatory particle, and upon this recognition, the 19S RP recruitsdeubiquitinases to remove the ubiquitin, unwinds the protein to limitits tertiary structure, and then shuttles it into the 20S CP. The 19S RPis also responsible for directly interacting with the N-termini of theα-rings of the 20S CP to open the pore or gate to allow proteinsubstrates to enter.³ It is the responsibility of the 20S CP tohydrolyze the protein into peptides that can then be recycled to makenew proteins or used as antigenic peptides. The 20S CP has the abilityto cleave proteins into peptides with chymotrypsin-, trypsin- andcaspase-like activity.

The 26S proteasome has been previously validated as a therapeutictarget. A number of small molecules have been developed that behave asproteasome inhibitors and lead to cytotoxicity in cancers.⁴ The mostwell-known is bortezomib (Velcade®), which is a covalent inhibitor thatinteracts with the chymotrypsin-like site on the 20S CP to preventprotein hydrolysis and is prescribed for patients with refractorymultiple myeloma. Recently, there have also been reports of inhibitorsto subunits that compose the 19S RP; however, none have made it into theclinic yet.^(5,6)

Hydrolysis of proteins is an essential process for all cell types, anddisruption of the activity of the proteasome, either the 26S or 20Sisoform, can lead to cell death. Unfortunately, as cells age or areaffected by protein accumulation diseases such as Parkinson's, the levelof proteins that assemble to form the 19S RP is greatly diminished ascompared to younger or healthy cells.⁷ Without the 19S RP,ubiquitin-dependent protein degradation cannot occur, but the 20S CP candegrade proteins, slowly, in an ubiquitin-independent manner.⁸ Thelevels of the proteins that compose the 20S CP remain the same betweendiseased and healthy cells.⁹ When the 20S CP is not capped by the 19SRP, only small, intrinsically disordered proteins can be degraded, butonly at a slow rate.

There has long been a general hypothesis that if one could increase therate at which the 20S CP can turn over proteins, this could alleviatethe negative effects associated with protein accumulation diseases.¹⁰⁻¹²It is believed that this can be done through two mechanisms with smallmolecules. The first is stimulating the gate of the 20S CP to open,allowing more substrates to enter at a faster rate.^(13,14) The secondmechanism is through an allosteric interaction with one of the activesites.¹⁵

There have been previous reports of peptides and small molecules thatcan stimulate the 20S CP. For example, peptides that mimic theC-terminus of some of the 19S RP's ATPases have been described tostimulate the 20S CP.¹⁶⁻¹⁸ There have also been natural productsreported to behave as 20S CP stimulation agents, including oleuropein, amajor component of olive oil, and betulinic acid, a natural productoriginally discovered from the white birch tree.¹⁹⁻²¹ The aforementionedmolecules were discovered by utilizing a small reporter peptide,composed of 3-4 amino acids plus a terminal aminomethyl coumarin group,FIG. 2A. Upon interaction with the 20S CP, the amino methyl coumaringroup is hydrolyzed from the peptide, and a fluorescent signal begins toaccumulate. For this to be a therapeutic route of interest, the 20S CPmust be stimulated to turn over a protein, and therefore, screening withsuch a small reporter peptide could yield results that are notbiologically relevant. Additionally, because of the small size of thereporter, it can easily diffuse into the 20S CP and be turned over withno stimulation. Performing a high throughput screen with a reporter thissmall will have a significant number of false negatives because of thehigh basal level of activity of the 20S CP, making the detection of weak20S CP stimulators a challenge, especially those that are allostericstimulators. There is a need to provide a more accurate reporter peptidethat can identify stimulators of 20S CP.

SUMMARY OF THE INVENTION

This disclosure provides a fluorescent reporter for identifying coreprotein (CP) 20S stimulators. The fluorescent reporter comprising thestructure of Lys(Y1)-Met-Ser-Gly-X-Ala-Ala-Thr-Ala-Glu(Y2)-Gly or a saltthereof, wherein X is selected from the group consisting of Phe, Arg andAsp:

wherein CT-L FRET, X=Phe (SEQ ID NO: 1); T-L FRET, X=Arg (SEQ ID NO: 2);and CP-L FRET, X=Asp (SEQ ID NO: 3).

This disclosure provides a group of compounds that stimulates CP 20S.The compounds have the structure named 1, 2 and 3. Among thesecompounds, compounds 2-3 provides gate opening function for 20S CP andcompound 1 provides allosteric interaction with beta-5 subunit of 20SCP, which stimulates chymotrypsin-like activity.

This disclosure further provides a method to identify a potent CP 20Sstimulator to accelerate protein degradation. The method comprising:

-   -   providing a fluorescence reporter with the following structure:    -   Lys(Y1)-Met-Ser-Gly-X-Ala-Thr-Ala-Glu(Y2)-Gly, wherein X is        selected from the group consisting of Phe, Arg and Asp;    -   Individually pairing the fluorescence reporter with each        candidate compound in a library and supplying CP 20S to the pair        to obtain the fluorescence reporter hydrolysis reading;    -   providing a positive control with the fluorescence reporter and        CP 20S stimulator to obtain a reference fluorescence reporter        hydrolysis reading;    -   Comparing each candidate compound associated fluorescence        reporter reading with the reference fluorescence reporter        hydrolysis reading; and    -   Identifying the compound that matches or surpasses the reference        fluorescence reporter hydrolysis reading as a hit compound        stimulator to CP 20S.

In some preferred embodiment, the aforementioned positive control issodium dodecyl sulfate (SDS) or AM-404.

In some preferred embodiment, the aforementioned fluorescence reporterhydrolysis reading is expressed by the rate of hydrolysis in relativefluorescence units (RFU) per minute (ΔRFU/Min).

This disclosure further provides a method of treating a proteinaccumulation related disease in a patient. The method comprising:

-   -   using a fluorescent reporter to identify at least one 20S CP        stimulator;    -   applying pharmaceutically effective amount of said at least one        20S CP stimulator to the patient.

In some preferred embodiment, the aforementioned fluorescent reporter isLys(Y1)-Met-Ser-Gly-X-Ala-Ala-Thr-Ala-Glu(Y2)-Gly, wherein X is selectedfrom the group consisting of Phe, Arg and Asp, wherein CT-L FRET, X=Phe(SEQ ID NO: 1); T-L FRET, X=Arg (SEQ ID NO: 2); and CP-L FRET, X=Asp(SEQ ID NO: 3).

In some preferred embodiment, the aforementioned disease is Parkinson'sdisease and the protein accumulated is α-synuclein.

These and other features, aspects and advantages of the presentinvention will become better understood with reference to the followingfigures, associated descriptions and claims.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

FIG. 1. The proteasome can degrade proteins through aubiquitin-dependent process when the 19S RP and 20S CP associate to formthe 26S proteasome. The 20S CP can degrade proteins but can only acceptsmall, intrinsically disordered proteins and does so at a slow rate.

FIG. 2. (FIG. 2A) Typically used reporter peptide to monitor thechymotrypsin-like activity of the 20S CP. (FIG. 2B) To more efficientlydetect 20S CP stimulation molecules, we developed a series of FRETprobes to monitor the variety of 20S CP activities.

FIG. 3. Accumulation of fluorescence upon the cleavage of the aminocoumarin group (FIG. 3A) or the CT-L FRET (FIG. 3B) reporter by the 20SCP with and without SDS. (FIG. 3C) Direct comparison of the basal levelof 20S CP activity to that when SDS is added with the four-amino acidreporter or the CT-L FRET. The rate of hydrolysis of the larger FRETreporter increases more in the presence of a stimulator than thefour-amino acid reporter.

FIG. 4. (FIG. 4A) Chemical structure of AM-404. (FIG. 4B) Increase influorescence intensity in the presence of AM-404 is dose-dependent. Theconcentrations of AM-404 had the following increases in the rate ofhydrolysis: 50.0 μM, 7-fold increase; 25.0 μM, 5-fold increase; 12.5 μM,4-fold increase.

FIG. 5. (FIG. 5A) Scatter plot of screening results utilizing the CT-LFRET reporter. The black dots represent the triplicates of the 20S CPbasal level, which was then normalized to zero for each plate ofcompounds. AM-404, a known 20S CP stimulator is the red dots which wereincluded as positive controls in the screening plates. The green dotsare the four hit molecules while the blue dots were not consideredfurther. (FIG. 5B) Four primary hit molecules that stimulated the 20S CPat least 50%.

FIG. 6. Dose response curves for 20S CP stimulation for the threeprimary hits. The EC50's listed here is the concentration of half of themaximum stimulation. Molecule 3 stimulates the 20S CP the most with 150%(FIG. 6C) over basal level followed by molecule 2 at 100% (FIG. 6B) andmolecule 3 at 50% (FIG. 6A).

FIG. 7. (FIG. 7A) Molecules 1-3 were tested for their ability tostimulate the 26S proteasome's CT-L activity. Only molecule 1 showed anystimulation ability. (FIG. 7B) The same molecules were tested with theiCP. The iCP has different active site subunits than the 20S CP but thegame gate subunits. In this case, molecules 2 and 3 showed an effect,while 1 did not.

FIG. 8. Comparison of the activity of the four primary hits with theCT-L FRET and the 4-amino acid reporter for detecting 20S CPstimulation. Molecules 2 and 3 would not have been considered primaryhits if we had utilized the smaller reporter.

FIG. 9. LC-MS analysis of the CT-L FRET reporter hydrolysis by the 20SCP.

FIG. 10. Rate of hydrolysis of the CT-L FRET reporter of the 20S CP atthe following concentrations of reporter: 2.5, 5, 10, 20, 40, 80, and160 μM.

FIGS. 11A and 11B.

FIG. 11A. Rate of hydrolysis of Suc-LLVY-AMC at the followingconcentrations of SDS: 0, 125, 250, 500, 1000, 2000, and 4000 μM.

FIG. 11B. Kinetic analysis of the addition of 500 μM or 1000 μM SDScompared to the basal activity of the 20S proteasome.

FIGS. 12A, 12B, 12C.

FIG. 12A. Increase in the rate of hydrolysis of Bz-VGR-AMC in thepresence of 500 μM SDS compared to the basal activity of the 20S CP:2-fold.

FIG. 12B. Increase in the rate of hydrolysis of T-L FRET reporter in thepresence of 500 μM SDS compared to the basal activity of the 20S CP:5-fold.

FIG. 12C. A succinct comparison of the rate of hydrolysis of the T-LFRET reporter and Bz-VGR-AMC in the presence of 500 μM SDS. The controlis the rate of hydrolysis by the 20S in the absence of a stimulator.

FIGS. 13A and 13B.

FIG. 13A Chemical structure of fenofibrate.

FIG. 13B Increase in fluorescence intensity in the presence offenofibrate compared to control.

FIG. 14. Decrease in the rate of hydrolysis of the CT-L FRET reporter inthe presence of 2 μM bortezomib: 40% decrease.

FIGS. 15A and 15B.

FIG. 15A. Kinetic analysis of the trypsin-like activity of the 20S CP inthe presence of 25 μM of compounds 1, 2, and 3.

FIG. 15B. A succinct comparison of the rate of hydrolysis of the T-LFRET reporter by the 20S CP in the presence of 25 μM of compounds 1, 2,and 3. The basal level is the rate of hydrolysis by the 20S CP in theabsence of a stimulator and is set to 100%.

FIGS. 16A and 16B.

FIG. 16A. Kinetic analysis of the caspase-like activity of the 20S CP inthe presence of 25 μM of compounds 1, 2, and 3.

FIG. 16B. A succinct comparison of the rate of hydrolysis of the CP-LFRET reporter by the 20S CP in the presence of 25 μM of compounds 1, 2,and 3. The basal level is the rate of hydrolysis by the 20S CP in theabsence of a stimulator and is set to 100%.

FIGS. 17A and 17B.

FIG. 17A. Kinetic analysis of the chymotrypsin-like activity of the 26Sproteasome in the presence of 25 μM of compounds 1, 2, and 3.

FIG. 17B. Kinetic analysis of the chymotrypsin-like activity of theimmunoproteasome core particle (iCP) in the presence of 25 μM ofcompounds 1, 2, and 3.

FIG. 18. MALDI analysis of the CT-L FRET and T-L FRET reporters.

TABLE S1 Rate and standard deviation values for the basal activity ofthe 20S CP, stimulated activity in the presence of AM-404 (positivecontrol), and activity in the presence of fenofibrate (negativecontrol). Sample: Mean Signal (Rate) Standard Deviation Basal(background) 5.300 0.1376 Positive Control 40.46 1.210 Negative Control5.795 0.1521

TABLE S2 Values obtained by inputting the data in Table S1 into theequations above. Standard Value S/N (pos) 255.5 S/B (pos) 7.634 % CV(pos) 2.991 % CV (neg) 2.625 Z′ 0.8821

DETAILED DESCRIPTION

While the concepts of the present disclosure are illustrated anddescribed in detail in the figures and the description herein, resultsin the figures and their description are to be considered as exemplaryand not restrictive in character; it being understood that only theillustrative embodiments are shown and described and that all changesand modifications that come within the spirit of the disclosure aredesired to be protected.

Unless defined otherwise, the scientific and technology nomenclatureshave the same meaning as commonly understood by a person in the ordinaryskill in the art pertaining to this disclosure.

To discover more biologically relevant 20S CP stimulating molecules andlimit the false negative rate during a high throughput screeningcampaign, we have developed fluorescence resonance energy transferreporters to monitor all three types of activity of the 20S CP, FIG. 2B.As we report here, these FRET reporters are over three-times moresensitive to stimulation than the traditionally utilized reporters. Weutilized the chymotrypsin-like FRET reporter (CT-L FRET) to screen alibrary of 800 small molecules and discovered three that stimulate the20S CP. Two molecules (compound were validated as 20S CP stimulatingprobes that elicit their effects through opening the gate, as bothmolecules increase the three types of 20S CP hydrolysis activity. Thethird molecule we present here, whose scaffold has been previouslyunreported to interact with the 20S CP, only stimulates thechymotrypsin-like activity of the 20S CP. This is the first time amolecule with this mechanism of action has been reported. This resulthighlights that our FRET reporters can discover both gate opening andallosteric stimulating probes.

The FRET reporters we present here have a number of advantages over thethree to four amino acid-coumarin reporters used previously. First, aswe demonstrated with the CT-L FRET reporter, it is 3.5-times moresensitive to stimulation of the 20S CP than Suc-LLVY-AMC, FIG. 3C. Thisis a major advantage when performing a high throughput screen ofthousands of molecules. Not only are false negatives of great concernduring screening, but so are false positives. Commonly, when performinga screen, primary hits will not be especially potent; therefore, it isessential to have a reporter sensitive enough to detect potential weakhits, limiting false negatives. When we screened 715 compounds with theCT-L FRET reporter, we detected four primary hits, giving a hit rate of0.6%.

As a proof of concept on the sensitivity of the CT-L FRET reporter, wealso tested these four primary hits with the Suc-LLVY-AMC reporter tosee if they would have been discovered. As shown in FIG. 8, molecules 1and 3 would not have been considered primary hits if this library ofmolecules was screened with the Suc-LLVY-AMC reporter. Not only willscreening with the CT-L FRET reporter be able to detect more moleculeswhen screening libraries previously untested for 20S CP stimulatingmolecules, it can also be used to re-screen libraries that wereanticipated to have hits but did not.

Our method presented here for screening for new 20S CP stimulatingmolecules has a distinct advantage over screening in cells. While incellulo screening does inherently report molecules that are able toenter the cell, the true biological target of the molecule can be veryunclear. This is especially true when screening for proteasomestimulators. The term proteasome stimulator has a very broad definition.For example, there are a number of molecules that have been reported asproteasome stimulators or enhancers but actually make no interactionwith the proteasome itself.^(23,32) These molecules do affect theability of the proteasome to degrade proteins but do so throughinteractions with deubiquitinases or proteasome-associated proteins.Additionally, current proteasome activity reporters cannot differentiatebetween the 20S CP and the 26S, and in the cell, both isoforms arepresent. While stimulating the 26S, either through ubiquitin-dependentor independent mechanisms, may be of interest, there is some cause forconcern. First, it was recently reported that stimulatingubiquitin-dependent proteasome activity can actually help cancer cellsto survive.³³ Second, as previously described, in diseased cells onlythe 20S CP is present, as the precursor proteins to form the 19S RP arenot produced at high enough levels. Therefore, in these diseases, onlythe 20S CP is available to degrade accumulated proteins, and moleculesthat directly affect this isoform have the greatest potential to betested as therapeutics. While 26S stimulating molecules may be ofinterest, screening for molecules should be focused on finding 20S CPstimulators, which cannot be done in a cell-based assay.

Concern has also been expressed on the toxic potential of molecules thatstimulate this protease. Obviously, stimulating the 20S CP could havenegative effects on cells by destroying proteins that are still requiredby the cell. However, we do not believe this will be the case. The 20SCP can only degrade small, intrinsically disordered proteins that arenot ubiquitinated. The amount of proteins that fall into this categoryis relatively small, but does include toxic proteins such as tau andα-synuclein.³⁴ Even upon stimulation, the 20S CP cannot degrade proteinssuch as GFP, GAPDH, or actin in a ubiquitin-independent manner.³⁵ Whilethere may be some toxicity associated with 20S CP stimulation, similarto what is observed with other drug mechanisms, based on the variety ofmolecules we have discovered here, we should be able to tailor theactivity to the amount desired based on the disease or target ofinterest.

Although we only screened a very small library, we discovered threecompounds that validated as 20S CP stimulators. Compound 3, which isvitamin E succinate, is an interesting result because of its long carbontail. There have been a number of other molecules with similar features,including SDS and AM-404, that are believed to be gate openers of the20S CP through a denaturing effect of the N-termini of the a-ring thatmakes up the gate. Compound 2, ursolic acid, is very similar instructure to betulinic acid, a previously reported 20S CP stimulator.²⁰Both of these compounds, because of their substantial activity in otherbiochemical assays, are not ideal to begin in-depth cellular analysis onthe effects of stimulating the 20S CP or animal experiments, but theirscaffolds represent a starting point for medicinal chemistry studies.Clearly, molecules with an aliphatic carbon chain, whose length andunsaturation has yet to be determined, can act as 20S CP gate openers.Without being bound by any theory, a similar hypothesis can be made forthe molecules with a steroid scaffold with stereochemistry andsubstitution similar to ursolic and betulinic acid. The triazinescaffold of molecule 1 is also an exciting medicinal chemistry avenue toexplore.³⁶ Based on a PubChem search, this molecule has not beenreported to be active in a significant amount of assays and is an easyscaffold to make a variety of derivatives to find more potent molecules.Synthesis and testing of derivatives of all three of these molecules maylead to other potent stimulators.

To begin our design of a more biologically relevant assay to discover20S CP stimulating molecules, we attempted to have a larger reporter toyield hit molecules that had more of a potential to stimulate the 20S CPto accept an intrinsically disordered protein. We also wanted to have areporter that was more challenging for the 20S CP to degrade without astimulator present. The small reporter most often used, FIG. 2A, tomonitor the chymotrypsin-like activity of the 20S CP can easily beturned over with no stimulating molecule present; because of this, ithas a very limited dynamic range, meaning that the difference betweenthe basal level of activity and stimulated activity of the 20S CP isvery small. There have been other proteasome activity reportersdeveloped, but these too are small peptides of three to four amino acidsin length and are not appropriate for stimulation studies.²² Reportersfor monitoring the activity of the proteasome in cells have also beendeveloped but cannot differentiate between activity of the 20S CP andthe 26S proteasome.²³⁻²⁵ There have also been gel- and NMR-based assaysfor detecting the activity of the 20S CP, but these are not amenable forhigh throughput screening.^(26,27)

In addition to designing a larger reporter, we also wanted to move thehydrolysis site away from the C-terminus to a more central position.This requires that more of the reporter needs to be shuttled into the20S CP, which would more accurately portray how a disordered proteinwould also fit in to be degraded. We wished to retain the speed and costeffectiveness of a fluorescent assay, which is why we ultimately decidedon developing FRET probes. There were report of FRET peptide designed tomonitor the activity of HIV protease, which uses the Dabcyl moiety asthe FRET acceptor and Edans as the donor.²⁸ We synthesized the largestpeptide we could to best mimic a disordered protein but still retainsufficient FRET efficiency to limit any background signal. The aminoacids between the FRET pairs were also carefully chosen so that only oneactive site type was engaged during a screen. Our first FRET reporterwas synthesized and tested to study the chymotrypsin-like activity ofthe 20S CP. At the fifth amino acid position is a phenylalanine, whichis recognized by the beta-5 subunit of the 20S CP. Hydrolysis of thisFRET reporter occurs at the C-terminal side of the phenylalanine(Supporting Information FIG. S1). This cleavage event then allows thesignal for Edans fluorescence to be obtained because it is no longerquenched by the Dabcyl moiety. Exchange of the phenylanine for anarginine or an aspartic acid allows one to monitor the trypsin- orcaspase-like activity, respectively. The remaining amino acids werechosen based on cost effectiveness, ease of coupling, and to aid inbuffer solubility. FIG. 2B shows the final reporter designs.

For all of our assays, we decided to monitor the 20S CP activity over 40minutes, taking a reading every two minutes to generate a rate ofhydrolysis (ΔRFU/min). While every attempt was made to keep the basallevel of the 20S CP constant, some loss of activity can occur for avariety of reasons from different batches. Monitoring the 20S CPactivity over a range of time to generate a rate, rather than a singletime point, minimizes the error from screen to screen. We next needed todetermine the amount of FRET reporter and 20S CP in each well to obtainsignificant signal. Previous reports have utilized 250 μM of theSuc-LLVY-AMC and between 1-10 nM of 20S CP to monitor the 20S CPactivity. The first test with the CT-L FRET we repeated the 250 μM ofreporter with 9 nM of 20S CP. Not surprisingly, this was too muchreporter and led to confusing results. The CT-L FRET reporter was thentested at decreasing concentrations keeping the concentration of 20S CPat 9 nM. Excitingly, the CT-L FRET reporter had the best response atmore than tenfold less (20 μM) than the Suc-LLVY-AMC probe, FIG. 10.

To determine the increased sensitivity to stimulation the CT-L FRETreporter provides over the smaller reporter, we tested both in thepresence of sodium dodecyl sulfate (SDS). SDS has long been considered a20S CP stimulator.²⁹ It is believed that it denatures the gate openingto allow more substrate to enter, leading to an increase in the amountof fluorescence observed. After determining the EC₅₀ of SDS (FIG. 11),we compared the basal level of activity of the 20S CP to that in thepresence of 500 μM of SDS with the CT-L FRET or the Suc-LLVY-AMCreporter. For this analysis, the basal level of the 20S CP was set to100% for both probes, and the percentage of increase in the rate offluorescent accumulation per minute in the presence of SDS wasdetermined. FIG. 3A shows the data obtained with the Suc-LLVY-AMCreporter. The average basal level of the 20S CP with this reporter wasdetermined to be 60 ΔRFU/min, while in the presence of SDS, the rateincreased to an average rate of 145 ΔRFU/min, a 2.4-fold increase. Theresult with the CT-L FRET is shown in FIG. 3B. With this reporter, theaverage basal level of the 20S CP was determined to be 2.9 ΔRFU/min, andafter treatment with SDS, the rate increased to an average rate of 24ΔRFU/min, an 8.3-fold increase in the rate.

The CT-L FRET is hydrolyzed at a much slower rate compared to theSuc-LLVY-AMC reporter. While this is significant, what is mostnoteworthy is the dramatic increase in the difference between basallevel and stimulated activities of the 20S CP, FIG. 3C. The stimulationthat SDS can induce is considered the maximum increase in activity the20S CP can exhibit by the gate opening mechanism. Therefore, the rangeof molecules our CT-L FRET reporter can detect during a high throughputscreening campaign is larger than that of the Suc-LLVY-AMC probe, as itis 3.5-fold more sensitive to stimulation. A similar result was observedwhen comparing the smaller trypsin-like reporter to our larger FRETreporter, FIG. 11.

To confirm that the CT-L FRET reporter could detect stimulators in adose response manner and that the result with SDS was not due to aninteraction between the CT-L FRET reporter and SDS, we also tested apreviously reported 20S CP stimulator.³⁰ AM-404, FIG. 4A, has beenidentified as a 20S CP stimulator with the Suc-LLVY-AMC with an EC₅₀≈32μM in cellulo. In a similar fashion as the SDS experiment, purified 20SCP was dosed with decreasing amounts of AM-404. As shown in FIG. 4B,AM-404 stimulates the 20S CP with the CT-L FRET reporter. At the lowestconcentration we tested, AM-404 stimulated the activity of the 20S CP4-fold. As a negative control, we tested fenofibrate, anotherhydrophobic molecule, with the CT-L FRET probe and the 20S CP. At avariety of doses, this molecule did not stimulate the 20S CP, FIG. 12.With all of these results in mind, we can conclude that the CT-L FRETreporter can effectively detect 20S CP stimulating molecules and do soin a dose-dependent manner.

We next wanted to utilize the CT-L FRET reporter to screen a library ofsmall molecules to find new 20S CP stimulating molecules. We screenedthe TimTec NPL-800 library, which is 800 natural products or naturalproduct derivatives. The library was first pre-screened at 25 μM with no20S CP over 40 minutes, looking for any change in fluorescence at theemission wavelength of Edans. Any molecule that showed a change insignal was excluded from screening. Additionally, this prescreen wasused to determine what background, if any, to remove from the screeningresults with the 20S CP. Next, fresh 96-well plates were prepared thatcontained 25 μM of the compound, 20 μM of the CT-L FRET reporter, and 9nM of 20S CP in a total volume of 50 μL. The plate was transferred to afluorescent plate reader at 37° C. and a reading was taken every twominutes over a 40 minute period. This was performed until all 715compounds were screened. 85 compounds were not screened with 20S CPbecause they did not pass our pre-screening qualifications. Wellscontaining 25 μM of AM-404, wells with no 20S CP, and wells to monitorthe basal level of the 20S CP were included in triplicate in everyplate. After all compounds were screened in singlet, the rates of 20S CPhydrolysis in the presence of each compound was calculated and comparedto the basal level, FIG. 5A and FIG. 13. We decided to select hits asthose that increased the rate of hydrolysis at least 50%. For cellexperiments, we did not want to dose with more than 25 μM, but a 50%increase in the biochemical assay should provide enough stimulation incells to see a desired signal. After this analysis, four compounds wereconsidered primary hits, FIG. 5B.

To validate these four compounds as 20S CP stimulating probes, weperformed a number of additional assays with newly purchased compound.First, the four hits were tested in triplicate at 25 μM, just as wasperformed in the primary screen. Molecules 4, Sennoside B, did notperform as it had in the initial screen. After liquidchromatography-mass spectrometry analysis of the compound from thescreening library and the new solid obtained, a significant amount ofthe non-glycosylated product was seen in the new stock. At this point,compound 4 was removed from the hit pool due to the difficulty inobtaining pure product to test and its overall poor drug-likeproperties. However, molecules 1-3 validated in triplicate at 25 μM.Since we had only ever tested the molecules at 25 μM, we chose toperform a dose response curve for all three. As shown in FIG. 6A-C, allthree compounds responded in a dose response manner. Molecule 3 is themost potent with an EC₅₀ of 7 μM, followed by compound 1 with an EC₅₀ of10 μM, and compound 2 with an EC₅₀ of 14 μM.

As previously mentioned the 20S CP has three types of active sites:chymotrypsin-, trypsin- and caspase-like. Until now, we have onlymonitored the increased activity of the chymotrypsin-like site of the20S CP. We wished to assess whether these three molecules are behavingas 20S CP gate openers or interacting more specifically with adesignated active site. If a molecule is increasing the hydrolysis rateof the 20S CP by opening the gate to allow more substrates to enter, theturnover rate of all three active sites should increase. If a compoundbehaves as an allosteric stimulator, only one active site should see anincrease in hydrolysis. To determine if our newly discovered moleculeselicit their stimulation effects either as a gate opener or allostericstimulator, we synthesized two additional FRET reporters. Thephenylalanine in the CT-L FRET reporter was exchanged with an arginineto monitor the trypsin-like activity (T-L FRET) or with an aspartic acidto evaluate the caspase-like activity (CP-L FRET) of the 20S CP.Molecules 1-3 were first tested with the T-L FRET reporter. At 25 μM, 2and 3 stimulated the trypsin-like activity of the 20S CP, 234% and 146%,respectively, FIG. 15. A similar result was obtained with the CP-L FRETreporter, FIG. 16. Most interestingly, 1 did not affect the trypsin- orcaspase-like activities of the 20S CP. From these results, we can inferthat 2 and 3 act as 20S CP gate openers, while 1 only increases thechymotrypsin-like activity of the 20S CP, potentially through anallosteric interaction with the beta-5 subunit. To the best of ourknowledge, this is the first example of a small molecule that directlyinteracts with the 20S CP to stimulate only the chymotrypsin-likeactivity.

As previously described, the 20S CP can be capped with the 19S RP toenable ubiquitin-dependent protein degradation. The 19S RP associatesdirectly with the alpha-ring of the 20S CP, opening the gate to allowsubstrates to enter. Molecules that behave as gate openers shouldtherefore have no effect on the 26S proteasome because the gate isalready open through the interaction with the 19S RP. This is indeed thecase with compounds 2 and 3, FIG. 7A. Neither molecules 2 or 3 affectedthe rate of the 26S proteasome, providing more evidence these moleculesbehave by opening the gate of the 20S CP. Molecule 1 does increase 26Sstimulation by 44%. This result further supports our mechanistichypothesis that 1 interacts with the beta-5 subunit as it stimulatesboth the 20S and 26S isoforms of the proteasome.

To further evaluate the mechanism of action of compounds 1-3, they weretested for their ability to stimulate the activity of theimmunoproteasome (iCP). The iCP and 20S CP contain the same proteinsubunits that form the gate, but the active site subunits are slightlydifferent. Interferon-gamma stimulates the production of the iCP activesites, which cleave proteins into peptides differently than the 20S CPdoes. The peptides produced by the iCP are typically more amenable to beloaded into an MHC-I complex. To support our hypothesis that compounds 2and 3 are gate openers, they were tested for their ability to stimulatethe iCP since the gate subunits are identical to the 20S CP. As shown inFIG. 7B compounds 2 and 3 do stimulate the chymotrypsin-like activity ofthe iCP significantly, while compound 1 shows essentially no increase inactivity. This result implies that molecule 1 has a specific interactionwith the beta-5 subunit of the 20S CP and less so with the beta-5isubunit of the iCP. The sequences of the beta-5 and the beta-5i subunitsare very similar with the exception of the residues surrounding theactive site threonine residue, called the S1 binding pocket.³¹ Thispotentially is where molecule 1 binds.

General Materials and Methods

Utilizing solid-phase peptide synthesis, the probes were synthesizedusing Fmoc-Gly-Wang resin, and the reactions were performed in frittedsyringes (purchased from Henke Sass Wolfe). Resin was purchased fromChem-Impex Int'l INC. F-moc protected natural amino acids were purchasedfrom Novabiochem. Fmoc-Glu(EDANS)-OH and Fmoc-Lys(Dabcyl)-OH werepurchased from Chem-Impex Int'l INC. COMU was purchased from Alfa Aesar.N,N-Diisopropylethylamine was purchased from Fisher Scientific.Piperidine was purchased from Sigma Aldrich. Dichloromethane andN,N-Dimethylformamide were purchased from Fisher Scientific.

Probe purification was performed on an Agilent HPLC, using a reversedphase column. Samples were detected using a UV lamp, lookingspecifically at 254 nm and 280 nm wavelengths. All sample analysis wasinitiated with an isocratic elution of 95% A at 14 mL/min for # minfollowed by a linear gradient of 5%-95% B at 14 mL/min over ## min, thenan isocratic elution for # min at ##% B, and re-equilibration with ##% Afor # min (A: H₂O, 0.1% formic acid; B: CH₃CN, 0.1% formic acid). Oncesamples were purified, fractions were collected, combined, andlyophilized, using SPECIFIC Lyophilizer.

Probe analysis was performed using MALDI imaging mass spectrometry.

Each probe was dissolved in dimethylsulfoxide (DMSO: for molecularbiology, purchased from Sigma Aldrich) and diluted using Tris-HCl (50mM, pH 7.7, purchased from Fisher Scientific). AM-404 was purchased fromTocris. Sodium dodecyl-sulfate was purchased from Alfa Aesar. Bortezomibwas purchased from INSERT NAME. Natural Products Library was purchasedfrom TimTec, LLC. Purified 20S Proteasome was purchased from Enzo LifeSciences, INC. Assays were performed with a 96-well plate (black, Cat.237108, ThermoFisher Scientific) using the Synergy 4 Plate Reader.

Probe Synthesis

To a 3 mL fitted syringe was added 49.9 mg (0.065 meq, 1 eq)Fmoc-Gly-Wang resin (0.658 meq/g). The resin was swelled in 2 mL 1:1DCM:DMF for 1 hr. The DCM:DMF was drained using apparatus. Each couplingof the amino acids was performed using 4 eq amino acid, 4 eq COMU, and 8eq DIPEA. The amino acid and COMU were dissolved in 1 mL DMF. DIPEA wasadded, and the solution was vortexed for 2 min before being added to theresin. Each coupling of the Fmoc-protected amino acids was performed for1 hr at 60° C. while shaking at 400 rpm. Following each coupling, theresin was rinsed six times: three times with 1 mL DMF followed by threewashes with 1mL DCM. After the final wash, the Kaiser test was performedto determine the completion of the coupling reaction. Once the couplingwas determined successful, the Fmoc was removed by adding 2 mL 20%piperidine in DMF to the resin in the reaction vessel, which was shakenat room temperature for 40 min. Following deprotection, the resin wasrinsed six times: three times with 1 mL DMF followed by three washeswith 1 mL DCM. After the final rinse, the Kaiser test was performed todetermine the completion of the deprotection. If the Kaiser test showedthe coupling or deprotection as unsuccessful, the reaction was repeatedunder the same conditions. The peptide was cleaved by adding a solutionof 1.9 mL trifluoroacetic acid, 500 μL DCM, and 500 μLtriisopropylsilane to the resin. The reaction vessel was shaken at roomtemperature for 2 hr. The solution was then allowed to drip into a 15 mLfalcon tube. The resin was rinsed three times with 1 mL DCM, and therinses were also allowed to drip into the 15 mL falcon tube. Thesolution was evaporated under a gentle stream of argon, leaving ared-colored residue.

To the residue was added 5 mL ether. The tube was vortexed for 1 min asthe peptide crashed out of solution. The solution was then centrifugedfor 1 min at 4700 rpm, and the ether was decanted. This was repeated twomore times. After the ether was decanted for the final time, a 16-gaugeneedle was used to make a small hole near the cap of the 15 mL falcontube, which was placed in a vacuum-sealed container to remove theremaining ether.

Probe Purification

The peptide was dissolved in 3 mL DMSO and 1 mL 1:1 DMSO:H₂O.Purification was performed using an Agilent HPLC.

All sample analysis was initiated with an isocratic elution of 95% A at14 mL/min for 3 min followed by a linear gradient of 5%-95% B at 14mL/min over 12 min, then an isocratic elution for 3 min at 95% B, andre-equilibration with ##% A for # min (A: H₂O, 0.1% trifluoroaceticacid; B: CH₃CN, 0.1% trifluoroacetic acid). Once samples were purified,fractions were collected, combined, and lyophilized, using SPECIFICLyophilizer.The mass of the lyophilized FRET reporter was determined tobe 5.1 mg, producing a 5% yield CT-L FRET Reporter Hydrolysis by the 20SCP

The lyophilized CT-L FRET reporter was dissolved in dimethylsulfoxide(for molecular biology) and diluted with Tris-HCl (50 mM, pH 7.7) tocreate a 10 μM CT-L FRET solution.

To verify that the CT-L FRET reporter was being hydrolyzed by thechymotrypsin-like activity of the 20S core particle (20S CP), thisreporter was incubated in triplicate with and without the 20S CP for 2hr at 37° C. The samples were prepared in a black 96-well plate thus: toeach well was added 40 μL 10 μM CT-L FRET (in Tris-HCl, 50 mM, pH 7.7).To the control wells was added 10 μL Tris-HCl. To the test wells wasadded 5 μL Tris-HCl and 5 μL 90 nM 20S CP in Tris-HCl (finalconcentration of 9 nM). The 96-well plate was incubated for 2 hr at 37°C. Following incubation, the control samples were combined in a 0.600 mLepitube. This was repeated for the test wells. ThermoScientific 100 μLC18 tips were then used to transfer the peptides in the control andsamples tubes from Tris-HCl into a 1:1 solution of H₂O:Acetonitrile with0.1% formic acid (FA). This was done following the procedure provided bythe manufacturer. Briefly, the samples were adjusted to 0.2%trifluoroacetic acid (TFA) by adding 15 μL 2.5% TFA to each sample. TheC18 tip was wet by aspirating then discarding 100 μL of 1:1H₂O:Acetonitrile. This was repeated once. The C18 tip was thenequilibrated by aspirating then discarding 100 μL 0.1% TFA in water.This was repeated once. To obtain the peptides, 100 μL of the controlsample was aspirate and dispensed 10 times. After the final aspiration,the sample volume was discarded. The C18 tip was rinsed by aspiratingand discarding 100 μL 0.1% TFA 95:5 H₂O:Acetonitrile. This was repeatedonce. The sample was eluted using 50 L 0.1% FA in 1:1 H₂O:Acetonitrile.This procedure was followed for both the control sample and the testsample. The elutions of these samples were then analyzed using theLC-MS. (FIG. 9).

Kinetic Screening Assay

Plate preparation for the kinetic assay was performed similarly for allassays. All 96-well plates contained the following in triplicate:reporter background, containing reporter only, and control wells,containing reporter and purified 20S CP. It was determined that eachwell will contain a total volume of 50 μL. To each well was added 40 μLof the coumarin or FRET reporters (in Tris-HCl, 50 mM, pH 7.7). To thereporter background wells was added 7.5 μL Tris-HCl and 2.5 μL of thesolvent in which the compound of interest was dissolved or diluted.Similarly, to the control wells was added 2.5 μL Tris-HCl and 2.5 μL ofthe solvent in which the compound of interest was dissolved or diluted.To the test wells was added 2.5 μL Tris-HCl and 2.5 μL of the compoundof interest at a predetermined concentration. Immediately prior torunning the assay on the Synergy 4 Plate Reader, 5 μL purified 20Sproteasome (90 nM in Tris-HCl) was added to the control and test wells.

The Synergy 4 Plate Reader was set to heat the plate at 37° C. and readfluorescence every 2 min over a 40 min period. The excitation andemission wavelengths were set according to the reporter. For the FRETreporter, the excitation and emission wavelengths were set to 335 nm and493 nm, respectively, corresponding to the excitation and emissionwavelengths of the Edans fluorophore. For the coumarin reporter, theexcitation and emission wavelengths were set to 380 nm and 460 nm,respectively.

Determining Concentration of FRET reporter for Kinetic Assay

The lyophilized CT-L FRET reporter was dissolved in 400 μLdimethylsulfoxide (for molecular biology), producing an 8127.3 μM stocksolution. This stock solution was used to make solutions of the FRETreporter at the following concentrations in Tris-HCl (50 mM, pH 7.7):160, 80, 40, 20, 10, 5, and 2.5 μM. The following concentrations wereverified using the Nano-drop, reading absorbance at 335 nm, theexcitation wavelength of Edans: 160, 80, 40, and 20 μM. The lowerconcentrations of FRET reporter had corresponding absorbance values toolow to be accurately measured by the Nano-drop.

These FRET reporter solutions were used in the kinetic assay in order todetermine the appropriate concentration of reporter to be used in ahigh-throughput screening campaign. For each concentration, backgroundand test wells were prepared in triplicate. The background wells wereprepared thus: to each well was added 40 μL FRET reporter and 10 μLTris-HCl (50 mM, pH 7.7). To each of the test wells was added 40 μL FRETreporter and 5 μL Tris-HCl. Immediately prior to performing the assay onthe Synergy 4 Plate Reader, 5 μL purified 20S proteasome (90 nM inTris-HCl ) was added to the test wells, totaling the well volume at 50μL (final concentration of 9 nM 20S proteasome). The Synergy 4 PlateReader was set to heat the plate at 37° C. and read fluorescence every10 min over a 2 hr period. The excitation and emission wavelengths wereset to 335 nm and 493 nm, respectively. The resulting data in relativefluorescence units (RFU) was analyzed using GraphPad Prism and wasplotted against time in minutes. The rate of hydrolysis (in ΔRFU/min) ofthe CT-L FRET reporter at each of the concentrations was plotted againstits corresponding concentration, FIG. 10. Based on the resulting curveand the inability to validate the concentrations of 10 μM CT-L FRETusing the Nano-drop, it was determined that 20 μM FRET reporter was theappropriate concentration to use for the screening kinetic assay.

Determining Concentration of SDS

Sodium dodecyl sulfate (SDS) was used to test the effect stimulating the20S proteasome has on the hydrolysis of the FRET reporters. Theappropriate concentration of SDS to use in the kinetic assay wasdetermined using the small chymotrypsin-like activity reporter,Suc-LLVY-AMC. For accurate comparison, the Suc-LLVY-AMC was prepared asa 20 μM solution in Tris-HCl (50 mM, pH 7.7). In triplicate, thefollowing final concentrations of SDS were analyzed for the effect eachhas on the activity of the 20S proteasome: 4000 μM, 2000 μM, 1000 μM,500 μM, 250 μM, 125 μM, and 0 μM, the basal activity of the 20Sproteasome. To each well was added 40 μL 20 μM reporter (in Tris-HCl)and 2.5 μL Tris-HCl. Solutions of SDS were prepared in Tris-HCl at thefollowing concentrations, corresponding to the final concentrations ofSDS aforementioned: 80 mM, 40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM, and 0 mMSDS, respectively. From these solutions, 2.5 μL was added to theappropriate wells in the 96-well plate. Immediately prior to reading theplate with the Synergy 4 plate reader, 5 μL purified 20S proteasome (90nM in Tris-HCl) was added to the wells at a final concentration of 9 nMper well.

The Synergy 4 Plate Reader was set to heat the plate at 37° C. and readfluorescence every 2 min over a 1 hr period. The excitation and emissionwavelengths were set to 380 nm and 460 nm, respectively, correspondingto the excitation and emission wavelengths of coumarin. The resultingdata in relative fluorescence units (RFU) was analyzed using GraphPadPrism and was plotted against time in minutes. The rate of hydrolysis(in ΔRFU/min) of the Suc-LLVY-AMC reporter at each of the concentrationsof SDS was plotted against its corresponding concentration, FIG. 11a .Based on the resulting curve and the hyperbolic curve obtained using1000 μM SDS (FIG. 11b ), it was determined that 500 μM SDS was theappropriate concentration to use to test the effect stimulating the 20Sproteasome has on the hydrolysis of the FRET reporters.

SDS Stimulation of the 20S CP

Stimulation of the 20S proteasome with SDS was determined using both thecoumarin and FRET reporters for the chymotrypsin-like and trypsin-likeactivities of the 20S CP. To each well was added 40 μL of the coumarinor FRET reporters (in Tris-HCl, 50 mM, pH 7.7). To the reporterbackground wells was added 10 μL Tris-HCl (50 mM, pH 7.7). Similarly, tothe control wells was added 5 μL Tris-HCl. To the test wells was added2.5 μL Tris-HCl and 2.5 μL 10 mM SDS in Tris-HCl. Immediately prior torunning the assay on the Synergy 4 Plate Reader, 5 μL purified 20Sproteasome (90 nM in Tris-HCl) was added to the control and test wells.The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

AM-404 Stimulation of the 20S CP

AM-404 was used as a positive control compound to test the dose-responsescreening capabilities of the CT-L FRET reporter. The followingconcentrations of AM-404 were tested in triplicate: 50, 25, and 12.5 μM.To each well was added 40 μL 20 μM CT-L FRET (in Tris-HCl, 50 mM, pH7.7). To the reporter background wells was added 5 μL Tris-HCl and 5 μL1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5 μL 1:1Tris-HCl:DMSO. To the 50 μM AM-404 test wells was added 2.5 μL Tris-HCland 2.5 μL 1000 μM AM-404 in DMSO. To the 25 μM AM-404 test wells wasadded 2.5 μL Tris-HCl and 2.5 μL 500 μM AM-404 in DMSO. To the 12.5 μMAM-404 test wells was added 2.5 μL Tris-HCl and 2.5 μL 250 μM AM-404 inDMSO. Immediately prior to running the assay on the Synergy 4 PlateReader, 5 μL purified 20S proteasome (90 nM in Tris-HCl) was added tothe control and test wells. The assay was performed using the Synergy 4plate reader as described for the general screening kinetic assayprocedure.

Effect of Fenofibrate on the 20S CP

Fenofibrate was used as a negative control compound to test thedose-response screening capabilities of the CT-L FRET reporter. Thefollowing concentrations of fenofibrate were tested in triplicate: 50,25, and 12.5 μM. To each well was added 40 L 20 μM CT-L FRET (inTris-HCl, 50 mM, pH 7.7). To the reporter background wells was added 5μL Tris-HCl and 5 μL 1:1 Tris-HCl:DMSO. Similarly, to the control wellswas added 5 μL 1:1 Tris-HCl:DMSO. To the 50 μM fenofibrate test wellswas added 2.5 μL Tris-HCl and 2.5 μL 1000 μM fenofibrate in DMSO. To the25 μM fenofibrate test wells was added 2.5 μL Tris-HCl and 2.5 μL 500 μMfenofibrate in DMSO. To the 12.5 μM fenofibrate test wells was added 2.5μL Tris-HCl and 2.5 μL 250 μM fenofibrate in DMSO. Immediately prior torunning the assay on the Synergy 4 Plate Reader, 5 μL purified 20Sproteasome (90 nM in Tris-HCl) was added to the control and test wells.The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Bortezomib Inhibition of the 20S CP

Bortezomib was used test the capabilities of the CT-L FRET reporter todetect inhibitors of the 20S CP. Bortezomib was tested at aconcentration of 2 μM in triplicate. To each well was added 40 μL 20 μMCT-L FRET (in Tris-HCl 50 mM, pH 7.7). To the reporter background wellswas added 5 μL Tris-HCl and 5 μL 1:1 Tris-HCl:DMSO. Similarly, to thecontrol wells was added 5 μL 1:1 Tris-HCl:DMSO. To the test wells wasadded 2.5 μL Tris-HCl and 2.5 μL 40 μM bortezomib in DMSO. Immediatelyprior to running the assay on the Synergy 4 Plate Reader, 5 μL purified20S proteasome (90 nM in Tris-HCl) was added to the control and testwells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Effect of 26S Stimulators on the 20S CP

Loperamide HCl and cyclosporin A have been shown to stimulate the 26Sproteasome at concentrations as low as 5 μM. This concentration was usedto test the effect of these compounds on hydrolysis of the CT-L FRET bythe 20S CP.

Each compound was tested at a concentration of 5μM in triplicate. Toeach well was added 40 μL 20 μM CT-L FRET (in Tris-HCl, 50 mM, pH 7.7).To the reporter background wells was added 5 μL Tris-HCl and 5 μL 1:1Tris-HCl:DMSO. Similarly, to the control wells was added 5 μL 1:1Tris-HCl:DMSO. To the loperamide HCl test wells was added 2.5 μLTris-HCl and 2.5 μL 100 μM loperamide HCl in DMSO. To the cyclosporin Atest wells was added 2.5 μL Tris-HCl and 2.5 μL 100 μM cyclosporin A inDMSO. Immediately prior to running the assay on the Synergy 4 PlateReader, 5 μL purified 20S proteasome (90 nM in Tris-HCl) was added tothe control and test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Screening Natural Product Library

The natural product library of 800 compounds was purchased as ten96-well plates of 80 compounds from TimTec LLC. Each compound had beendissolved in 50 μL of DMSO to produce a 10 mM solution of compound. Adiluted stock of each plate was made by pipetting 4 μL of 10 mM compoundinto 76 μL DMSO in a corresponding 96-well plate. Then, 80 μL Tris-HCl(50 mM, pH 7.7) was added to produce a final concentration of 250 μM ofcompound in 1:1 DMSO:Tris-HCl.

Due to the 96-well vessel containing 80 compounds in stock solutions, 80compounds were screened in each 96-well plate. Prior to performing thescreen with the 20S CP, background plates were prepared and analyzed in96-well plates for the compounds of interest. To these wells was added45 μL Tris-HCl and 5 μL of the compound of interest at 250 μM in 1:1Tris-HCl:DMSO, generating 25 μM a final concentration of compound ineach well. The assay was then performed using the Synergy 4 plate readeras described for the general screening kinetic assay procedure. This wasdone in order to determine the amount of background fluorescenceexhibited by each compound. Those compounds exhibiting a significantchange in fluorescence signal over time were excluded from screening.

The remaining compounds were then screened in singlet with the 20S CP;however, each plate was screened with the following in triplicate: CT-LFRET background, CT-L FRET control (containing reporter and purified 20SCP), and a positive control (containing reporter, purified 20S CP, and25 μM AM-404). To each well was added 40 μL 20 μM reporter (inTris-HCl). To the reporter background wells was added 5 μL Tris-HCl and5 μL 1:1 Tris-HCl:DMSO. Similarly, to the controls wells was added 5 μL1:1 Tris-HCl:DMSO. To the test wells was added 5 μL of the compound ofinterest at 250 μM in 1:1 Tris-HCl:DMSO (final concentration of 25 μM).Immediately prior to running the assay on the Synergy 4 Plate Reader, 5μL purified 20S proteasome (90 nM in Tris-HCl) was added to the controland test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Validating Hit Compounds in Triplicate

A 500 μM solution was made for each of the hit compounds (1, 2, 3, and4) in DMSO by diluting the 10 mM stock solution purchased from TimTec,LLC. The stimulation capabilities of the compounds were then tested intriplicate using the CT-L FRET reporter.

To each well was added 40 μL 20 μM reporter (in Tris-HCl). To thereporter background wells was added 5 μL Tris-HCl and 5 μL 1:1Tris-HCl:DMSO. Similarly, to the controls wells was added 5 μL 1:1Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and 2.5 μL ofthe 500 μM solution of the compound of interest in DMSO (finalconcentration of 25 μM). Immediately prior to running the assay on theSynergy 4 Plate Reader, 5 μL purified 20S proteasome (90 nM in Tris-HCl)was added to the control and test wells.

Background wells were prepared in singlet for the compounds of interest.To these wells was added 47.5 μL Tris-HCl and 2.5 μL of the 500 μMsolution of the compound of interest in DMSO.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Once validated, it was discovered that compounds 2, 3, and 4 could bepurchased more cheaply from other sources. These compounds were boughtfrom Sigma Aldrich and then tested in the manner previously stated.

Screening Hit Compounds with Suc-LLVY-AMC

Similarly, a 500 μM solution was made for each of the hit compounds (1,2, 3, and 4) in DMSO by diluting the 10 mM stock solution purchased fromTimTec, LLC. The stimulation capabilities of the compounds were thentested in triplicate using the Suc-LLVY-AMC reporter.

To each well was added 40 μL 20 μM reporter (in Tris-HCl). To thereporter background wells was added 5 μL Tris-HCl and 5 1:1Tris-HCl:DMSO. Similarly, to the controls wells was added 5 μL 1:1Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and 2.5 μL ofthe 500 μM solution of the compound of interest in DMSO (finalconcentration of 25 μM). Immediately prior to running the assay on theSynergy 4 Plate Reader, 5 μL purified 20S proteasome (90 nM in Tris-HCl)was added to the control and test wells.

Background wells were prepared in singlet for the compounds of interest.To these wells was added 47.5 μL Tris-HCl and 2.5 μL of the 500 μMsolution of the compound of interest in DMSO.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Screening 1, 2, and 3 with T-L FRET

The ability of compounds 1, 2, and 3 to stimulate the trypsin-likeactivity of the 20S CP was tested by screening these compounds intriplicate using the T-L FRET reporter.

To each well was added 40 μL 20 μM T-L FRET reporter (in Tris-HCl, 50mM, pH 7.7). To the reporter background wells was added 5 μL Tris-HCland 5 μL 1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5μL 1:1 Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and2.5 μL of a 500 μM solution of the compound of interest in DMSO. Thisresulted in a final concentration of 25 μM of the compound in the96-well plate. Immediately prior to running the assay on the Synergy 4Plate Reader, 5 μL purified 20S proteasome (90 nM in Tris-HCl) was addedto the control and test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Screening 1, 2, and 3 with T-L FRET

The ability of compounds 1, 2, and 3 to stimulate the caspase-likeactivity of the 20S CP was tested by screening these compounds intriplicate using the CP-L FRET reporter.

To each well was added 40μL 20 μM CP-L FRET reporter (in Tris-HCl, 50mM, pH 7.7). To the reporter background wells was added 5 μL Tris-HCland 5 μL 1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5μL 1:1 Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and2.5 μL of a 500 μM solution of the compound of interest in DMSO. Thisresulted in a final concentration of 25 μM of the compound in the96-well plate. Immediately prior to running the assay on the Synergy 4Plate Reader, 5 μL purified 20S proteasome (90 nM in Tris-HCl) was addedto the control and test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Dose Response Assay for 1, 2, and 3

The ability of compounds 1, 2, and 3 to stimulate the CT-L activity ofthe 20S proteasome was tested in a dose-response manner at the followingconcentrations of compound in triplicate: 400, 200, 100, 50, 25, 12.5,6.25, 3.125, 1.5625, 0.78125 and OW.

To each well was added 40 μL 20 μM reporter (in Tris-HCl) and 2.5 μLTris-HCl. To the reporter background wells was added 5 μL Tris-HCl and 5μL 1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5 μL 1:1Tris-HCl:DMSO. To the test wells was added 2.5 μIL Tris-HCl. Solutionsof 1 were prepared in DMSO at the following concentrations,corresponding to the final concentrations of 1 mentioned above: 8000,4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μM 1,respectively. From these solutions, 2.5 μL was added to the appropriatetest wells in the 96-well plate. Immediately prior to reading the platewith the Synergy 4 plate reader, 5 μL purified 20S proteasome (90 nM inTris-HCl) was added to the wells at a final concentration of 9 nM perwell.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

The above procedure was also performed for 2 and 3.Screening 1, 2, and 3 with 26S Proteasome

Compounds 1, 2, and 3 were screened with the 26S proteasome intriplicate using the CT-L FRET reporter.

To each well was added 40 μL 20 μM CT-L FRET reporter (in Tris-HCl, 50mM, pH 7.7). To the reporter background wells was added 5 μL Tris-HCland 5 μL 1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5μL 1:1 Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and2.5 μL of a 500 μM solution of the compound of interest in DMSO. Thisresulted in a final concentration of 25 μM of the compound in the96-well plate. Immediately prior to running the assay on the Synergy 4Plate Reader, 5 μL purified 26S proteasome (62.5 μg/mL in Tris-HCl) wasadded to the control and test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Screening 1, 2, and 3 with the Immunoproteasome

Compounds 1, 2, and 3 were screened with the immunoproteasome intriplicate using the CT-L FRET reporter.

To each well was added 40 μL 20 μM CT-L FRET reporter (in Tris-HCl, 50mM, pH 7.7). To the reporter background wells was added 5 μL Tris-HCland 5 μL 1:1 Tris-HCl:DMSO. Similarly, to the control wells was added 5μL 1:1 Tris-HCl:DMSO. To the test wells was added 2.5 μL Tris-HCl and2.5 μL of a 500 μM solution of the compound of interest in DMSO. Thisresulted in a final concentration of 25 μM of the compound in the96-well plate. Immediately prior to running the assay on the Synergy 4Plate Reader, 5μL purified 26S proteasome (62.5 μg/mL in Tris-HCl) wasadded to the control and test wells.

The assay was performed using the Synergy 4 plate reader as describedfor the general screening kinetic assay procedure.

Kinetic Assay Data Analysis

All of the data obtained from the Synergy 4 plate reader for the kineticscreening assay was processed using GraphPad Prism. With the exceptionof the data obtained from screening the Natural Product Library, all ofthe raw data obtained was directly analyzed with GraphPad Prism. For theNatural Product Library, the raw data obtained from the background wellswas subtracted from the raw data obtained from the test wells. Theresulting data was considered normalized; the raw data from the controlwells and the normalized data from the test compounds were analyzedusing the GraphPad Prism program.

For all of the assays, the fluorescent data was plotted against time inminutes. The linearized slope produced by these plots was considered therate of hydrolysis of the fluorescent reporters by the 20S proteasome.

GraphPad Prism was used to produce the dose-response plots of the CT-LFRET reporter (FIG. 10), SDS (FIGS. 11a ), 1, 2, and 3.

Appendix High-Throughput Screen Calculations

The data obtained from testing the effects of the positive control(AM-404) and the negative control (fenofibrate) on the 20S CP using theCT-L FRET reporter were used to determine the efficiency of using theCT-L FRET reporter in a HTS campaign. The following equations were used:

${{Signal}\text{-}{to}\text{-}{noise}\mspace{14mu}{{ratio}\left( {S\text{/}N} \right)}\text{:}\mspace{14mu}\frac{S}{N}} = \frac{{{mean}\mspace{14mu}{signal}} - {{mean}\mspace{14mu}{background}}}{{standard}\mspace{14mu}{deviation}\mspace{14mu}{of}\mspace{14mu}{background}}$${{Signal}\text{-}{to}\text{-}{background}\mspace{14mu}{{ratio}\left( {S\text{/}B} \right)}\text{:}\mspace{14mu}\frac{S}{B}} = \frac{{mean}\mspace{14mu}{signal}}{{mean}\mspace{14mu}{background}}$${{Coefficient}\mspace{14mu}{of}\mspace{14mu}{{variation}\left( {\%\mspace{14mu}{CV}} \right)}\text{:}\mspace{14mu}\%\mspace{14mu}{CV}} = {100*\frac{{standard}\mspace{14mu}{deviation}}{{mean}\mspace{14mu}{signal}}}$${Z^{\prime}\text{-}{{factor}\left( Z^{\prime} \right)}\text{:}\mspace{14mu} Z^{\prime}} = {1 - \frac{3\left( {{{SD}\mspace{14mu}{pos}\mspace{14mu}{control}} - {{SD}\mspace{14mu}{neg}\mspace{14mu}{control}}} \right)}{{{{mean}\mspace{14mu}{pos}\mspace{14mu}{control}} - {{mean}\mspace{14mu}{neg}\mspace{14mu}{control}}}}}$

In conclusion, we report here a new set of FRET reporters to screen for20S CP stimulating molecules. We show that these reporters are much moresensitive to stimulation than the commonly used three to four aminoacid-coumarin reporters but still retain the high throughput nature of afluorescent assay. With this added sensitivity, we for the first timereport here a small molecule that elicits only stimulation of thechymotrypsin-like activity of the 20S CP. We also discovered two othermolecules that present as 20S CP gate openers. Molecules that stimulatethe activity of the 20S CP through either of these mechanisms are ofgreat interest to evaluate the validity of this therapeutic strategy fortreating protein-accumulation diseases. Stimulation of the 20S CP hasbeen proposed as a potential therapy for Parkinson's disease, Alzheimer's disease, and to limit some negative effects associated with aging.³⁷The same mechanism of 20S CP stimulation may not be applicable to all ofthese disease states, and with the molecules we have in hand, we canbegin to elucidate which mechanism is applicable for which disease type.The scaffolds we discovered can be modified to generate more potent andselective 20S CP stimulators to begin to evaluate the therapeuticpotential of this mechanism in 3-D cell cultures of neurons, senescentcell models, and eventually animal models of these diseases. Finally, wehope these new FRET reporters are used by others to continue to discovermore 20S CP stimulation molecules to aid in the further exploration ofthis potential therapeutic mechanism.

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1. An assay kit for identifying a regulator for core protein 20S (20SCP) comprising a fluorescent reporter and a positive control, whereinsaid fluorescent reporter having a structure of

or a salt thereof, wherein CT-L FRET, X=Phe (SEQ ID NO: 1); T-L FRET,X=Arg (SEQ ID NO: 2); and CP-L FRET, X=Asp (SEQ ID NO: 3).
 2. The assaykit according to claim 1, wherein the positive control is sodium dodecylsulfate (SDS) or AM-404.
 3. A method of treating a protein accumulationrelated disease in a patient comprising the step of administrating a 20SCP stimulator together with one or more excipients to the patient inneed of relief from said disease, wherein said CP 20S comprises

or a salt thereof.
 4. The method of claim 3, wherein compounds 2-3provide gate opening function for 20S CP.
 5. The method of claim 3,wherein compound 1 provides allosteric interaction with beta-5 subunitof 20S CP and stimulates chymotrypsin-like activity.
 6. The methodaccording to claim 3, wherein said disease is Parkinson's disease andthe accumulated protein is α-synuclein.